The accurate segregation of the duplicated genome into daughter cells at mitosis and meiosis is a critically important component of the normal growth and differentiation of multicellular organisms. Improper chromosome segregation is directly linked to human developmental disorders, notably Down's syndrome. The work proposed here is designed to elucidate in detail the molecular components of the human kinetochore, the chromosomal structure that is directly responsible for linking chromosomes to the mitotic segregation apparatus. Autoimmune disease sera from patients suffering CREST scleroderma have been shown to recognize three specific kinetochore binding proteins present on human chromosomes, termed CENP-A, CENP-C. One major goal of this work, already partially realized, is to use CREST autoantibodies to isolated cDNA clones encoding the CENPs using the techniques of expression vector cloning. The primary structures of these proteins will be determined by DNA sequencing, potentially providing insight into kinetochore structure and function. CENP protein expression techniques will be developed for use in direct analysis of CENP:DNA interactions and to generate defined immmunological reagents for refined analysis of CENP function. A second major goal is to use anti-CENP antibodies to directly isolate kinetochore particles. DNA isolated from purified kinetochores (kDNA) will be used to establish recombinant DNA libraries for the cloning and analysis of these important sequences. Assays will be developed to analyze CENP:DNA binding interactions in vitro and the structure of kDNA:CENP- containing chromatin will be investigated in vitro and in vivo using Xenopus oocyte assay systems. Kinetochore structure and function will be probed in mammalian cells by re-introduction of CENP coding sequences microinjection of transfection to disrupt or abolish normal CENP expression, analyzing the altered mitotic phenotypes of cells. The role of kDNA sequences in chromosome segregation will be explored by developing systems for reconstitution of kinetochore function on extrachromosomal plasmids and by potentially disruptive amplification of kDNA. These experiments will establish tools and methods for detailed molecular analysis of the mechanisms of human chromosome segregation.